1. The genetic basis for the development of cancer

2. Two classes of genes are targets for the mutations:
Cancers arise through a multistage process in which inherited and somatic mutations of cellular genes lead to clonal selection of variant progeny with the most robust and aggressive growth properties.
Two classes of genes are targets for the mutations:
Protooncogenes
tumor-suppressor genes

3. A small fraction of all mutations in cancer cells are constitutional
The vast majority of the mutations that contribute to the development and behavior of cancer cells
are somatic (ie, arising during tumor development)
present only in the neoplastic cells of the patient
A small fraction of all mutations in cancer cells are constitutional
present in all somatic cells of affected individuals
such mutations not only predispose to cancer, but can also be passed on to future generations.

4. Tumor Suppressor Genes

5. A large number of tumor suppressor genes have been hypothesized to exist
Thus far, approximately 20 tumor-suppressor genes have been identified and definitively implicated in cancer development.
The cellular functions of the tumor-suppressor genes appear to be diverse

6. Cancer-inducing genes, specifically viral oncogenes, act in a dominant fashion
Viral oncogenes dictate cellular behavior in spite of the continued presence and expression of opposing cellular genes within the virus-infected cell that usually functioned to ensure normal cell proliferation.
The viral genes were able to induce a dominant phenotype—they were bringing about a cell transformation.
Most human cancers do not seem to arise as consequences of tumor virus infections

7. Cell fusion experiments indicate that the cancer phenotype is recessive
Figure 7.1 Experimental fusion of
cells When cells growing adjacent to
one another in monolayer culture are
exposed to a fusogenic agent, such as
inactivated Sendai virus or polyethylene
glycol (PEG), they initially form a
heterokaryon with multiple nuclei;
formation of such a cell with two nuclei
is shown here. When this cell
subsequently passes through mitosis, the
two sets of parental chromosomes are
pooled in a single nucleus. During
propagation of the resulting tetraploid
cell in culture, the descendant cells often
shed some of these chromosomes,
thereby reducing their chromosome
complement to a quasi-triploid or
hyperdiploid state (not shown).
Figure 7.2 Appearance of
experimentally fused cells Typical
initial products of fusing cultured cells
using a fusogenic agent (Figure 7.1; in
this case inactivated Sendai virus) are
seen here. Use of selection media and
selectable marker genes can ensure the
survival of a bi- or multinucleated cell
carrying nuclei deriving from two distinct
parental cell types. Conversely, use of
these marker genes ensures the
elimination of fused cells whose nuclei
derive from only one or the other
parental cell type. If such a hybrid cell is
viable, the complements of
chromosomes are intermingled in a
single nucleus during the subsequent
mitosis. (A) In this image, radiolabeled
mouse NIH 3T3 cells have been fused
with monkey kidney cells, resulting in a
cell with two distinct nuclei. The (larger)
mouse nucleus is identified by the silver
grains that were formed during
subsequent autoradiography.
(B) Polykaryons may form with equal or
greater frequency following such fusions
but are usually unable to proliferate and
spawn progeny. This polykaryon contains
nine nuclei (arrows). (Courtesy of
S. Rozenblatt.)

8. fusion of a cancer cell with a wild-type cell
The resulting hybrid cells have lost the ability to form tumors when these hybrid cells were injected into appropriate host animals.
This, unexpectedly, mean that the malignant cell phenotype is recessive to the phenotype of normal, wild-type growth.
Exception: when the transformed parental cell had been transformed by tumor virus infection.

9. Tumor suppressor genes (TSGs) Hypothesis
Normal cells carry genes that constrain or suppress their proliferation.
During the development of a tumor, the evolving cancer cells inactivate one or more of these genes.
Once these growth-suppressing genes are lost, the proliferation of the cancer cells accelerates.
As long as the cancer cell lacks these genes, it continues to proliferate in a malignant fashion.
When wild-type, intact versions of these genes operate once again within the cancer cell (by cell fusion) it will loose its ability to proliferate or to form tumors.

10. The retinoblastoma tumor is the first example of tumor suppressor genes
Sporadic form:
unilateral and unifocal
once the tumor is eliminated, no further risk
Familial form
bilateral and often multi-focal
curing the eye tumor does not protect the children from a greatly increased risk to bone cancers and other cancers

11. Familial form
The familial form of retinoblastoma is passed from one generation to the next in a fashion that conforms to the behavior of a Mendelian dominant allele.
Figure 7.5 Unilateral versus bilateral
retinoblastoma (A) Children with
unilateral retinoblastoma and without an
afflicted parent are considered to have a
sporadic form of the disease, while those
with bilateral retinoblastoma suffer from
a familial form of this disease. In this
graph, the clinical courses of a group of
1601 retinoblastoma patients who had
been diagnosed between 1914 and
1984 were followed. As is apparent,
those cured of bilateral tumors (red line)
have a dramatically higher risk of
developing second (and subsequently
occurring tumors) in a variety of organ
sites than those with unilateral tumors
(blue line). [A portion of this elevated
risk is attributable to tumors that arose
in the vicinity of the eyes because of the
radiotherapy (i.e. treatment by X-ray
irradiation) that was used to eliminate
the retinoblastomas when these
individuals were young.] (B) This
pedigree shows multiple generations of
a kindred afflicted with familial
retinoblastoma, a disease that usually
strikes only 1 in 20,000 children. Such
multiple-generation pedigrees were
rarely observed before the advent of
modern medicine, which allows an
affected child to be cured of the disease
and therefore reach reproductive age.
Males (squares), females (circles),
affected individuals (green filled circles,
squares), unaffected individuals (open
circles, squares). (A, from
R.A. Kleinerman, M.A. Tucker,
R.E. Tarone et al., J. Clin. Oncol.
23:2272–2279, 2005; B, courtesy of
T.P. Dryja.)
Figure 7.5b The Biology of Cancer (© Garland Science 2007)

12. Kinetics of Rb: familial (bilateral) vs. sporadic (unilateral)
the rate of appearance of familial tumors was consistent with a single random event (mutation)
the sporadic tumors behaved as if two random events were required
Figure 7.6 Kinetics of appearance of unilateral and bilateral
retinoblastomas Alfred Knudson Jr. studied the kinetics with which
bilateral and unilateral retinoblastomas appeared in children. He
calculated that the bilateral cases (tumors in both eyes) arose with onehit
kinetics, whereas the unilateral tumors (affecting only one or the
other eye) arose with two-hit kinetics. Each of these hits was presumed
to represent a somatic mutation. The fact that the two-hit kinetics
involved two copies of the Rb gene was realized only later. Percentage of
cases not yet diagnosed (ordinate) at age (in months) indicated (abscissa).
(From A.G. Knudson Jr., Proc. Natl. Acad. Sci. USA 68:820–823,1971.)

13. Two-hit hypothesis
Two “hits” or mutagenic events were necessary for retinoblastoma development
In an individual with the inherited form:
the first hit is present in the germ line, and thus in all cells of the body.
a second somatic mutation was hypothesized to be necessary for promoting tumor formation.
The second mutation explain the behavior of a Mendelian dominant allele.
In the nonhereditary form:
both mutations were proposed to arise somatically within the same cell.

14. Two-hit hypothesis
Each of the two hits could theoretically be in different genes
Subsequent studies led to the conclusion that both hits were at the same genetic locus, ultimately inactivating both alleles of the retinoblastoma (RB1) susceptibility gene
Figure 7.7 Dynamics of retinoblastoma
formation The two affected
genes predicted by Knudson (see Figure
7.6) are the two copies of the Rb gene
on human Chromosome 13. In familial
retinoblastoma, the zygote (fertilized
egg) from which a child derives carries
one defective copy of the Rb gene, and
all retinal cells in this child will therefore
carry only a single functional Rb gene
copy. If this surviving copy of the Rb
gene is eliminated in a retinal cell by a
somatic mutation, that cell will lack all
Rb gene function and will be poised to
proliferate into a mass of tumor cells. In
sporadic retinoblastoma, the zygote is
genetically wild type at the Rb locus. In
the retina of a resulting child, retinoblastoma
development will require two
successive somatic mutations striking the
two copies of the Rb gene carried by a
lineage of retinal precursor cells, yielding
once again the same outcome—a cell
poised to proliferate into a tumor mass.
Because only a single somatic mutation
is needed to eliminate Rb function in
familial cases, multiple cells in both eyes
are affected. However, the two somatic
mutations required in sporadic disease
are unlikely to affect a single cell lineage,
yielding at most one tumor.

15. Loss of Rb heterozygosoty (LOH) Mitotic recombination: a possible mechanism to eliminate the wild-type copy of Rb gene
The probability of inactivating a single gene copy by mutation is on the order of 10-6 per cell generation
The probability of silencing both copies is on the order of per cell generation.
It seems highly unlikely that both copies of the Rb gene could be eliminated through two recessive mutational event in the relatively small target cell populations in the developing retina (about 106 cells).

16. Loss of Rb heterozygosoty (LOH) Mitotic recombination: a possible mechanism to eliminate the wild-type copy of Rb gene
This mitotic recombination was found to occur at a frequency of 10–5 to 10–4 per cell generation
easier way for a cell to rid itself of the remaining wild-type copy of the Rb gene than mutational inactivation of this gene copy, which, as mentioned above, was known to occur at a frequency of about 10–6 per cell generation.
Figure 7.8 Elimination of wild-type
Rb gene copies Mitotic recombination
can lead to loss of heterozygosity (LOH)
of a gene such as Rb. Genetic material is
exchanged between two homologous
chromosomes (e.g., the two
Chromosomes 13) through the process
of genetic crossing over, occurring in the
G2 phase or, less often, the M phase of
the cell cycle. Once the crossing over has
occurred, the subsequent segregation of
chromatids may yield a pair of daughter
cells both of which retain heterozygosity
at the Rb locus. With equal probability,
this process can yield two daughter cells
that have undergone LOH at the Rb
locus (and other loci on the same
chromosomal arm), one of which is
homozygous mutant at the Rb locus
while the other is homozygous wild type
at this locus.

17. Loss of Rb heterozygosoty (LOH) Gene conversion: another possible mechanism to eliminate the wild-type copy of Rb gene
When gene conversion involve copying of an already inactivated Rb allele, for example, then once again LOH will have occurred in this chromosomal region.
known to occur
even more
frequently per cell
generation than
does mitotic
recombination.
Figure 7.9 Gene conversion During
the process of gene conversion, DNA
polymerases initially begin to use a
strand on one chromosome (red) as a
template for the synthesis of a new
daughter strand of DNA (blue). After
advancing some distance down this
template strand, the polymerase may
continue replication by jumping to the
homologous chromosome and using a
DNA strand of this other chromosome
(green) as a template for the continued
elongation of the daughter strand. After
a while, the polymerase may jump back
to the originally used DNA template
strand and continue replication. In this
manner, a mutant tumor suppressor
gene allele, such as a mutant allele of
Rb, may be transmitted from one
chromosome to its homolog, replacing
the wild-type allele residing there.

18. The Rb gene often undergoes loss of heterozygosity in tumors
In a small number of retinal tumors, careful karyotypic revealed interstitial deletions within the long (“q”) arm of Chromosome 13.
All of these deletions caused the loss of chromosomal material in the 4th band of the 1st region of this chromosomal arm (13q14).

19. A number of genes (including Rb) in this region had been lost simultaneously by the developing retinal tumor cells.
This is precisely the outcome predicted by the tumor suppressor gene theory.
LOH achieved by simply breaking off and discarding an entire chromosomal region without replacing it with a copy duplicated from the other, homologous chromosome is called hemizygosity
Figure 7.10 Chromosomal localization of the Rb locus (A) The
presence of distinct darkly staining bands in human chromosomes made
it possible to delineate specific subregions in each of the chromosomes
(above). In this case, careful study of the karyotype of retinoblastoma
cells, performed on the condensed chromosomes of late prophase or
metaphase cells, revealed the presence of a deletion affecting the long
arm (q) of Chromosome 13, in a 6-year-old retinoblastoma patient
(arrow, left), while Chromosomes 14 and 15 appeared normal.
(B) Careful cytogenetic analysis revealed that the deletion was localized
between bands 13q12 and q14 (below), that is, between the 2nd and
4th bands of the 1st region of the long (q) arm of Chromosome 13.
(A, from U. Francke, Cytogenet. Cell Genet. 16:131–134, 1976.)

20. Mutations of the Rb gene
Figure 7.12 Mutations of the Rb gene
The cloned Rb cDNA could be used as a
probe in Southern blot analyses of
genomic DNAs from a variety of
retinoblastomas and an osteosarcoma.
Each of the 10 exons of the Rb gene
(blue cylinders) is labeled by its length in
kilobases. The normal (N) version of the
gene encompasses a chromosomal region
of approximately 190 kilobases. However,
analysis of a subset of retinoblastoma
DNAs indicated that significant portions
of this gene had suffered deletion. The
beginning and end points of these
deletions are indicated by brackets. Thus,
tumors 41 and 9 lost the entire Rb gene,
apparently together with flanking
chromosomal DNA segments on both
sides. Tumors 44, 28, and 3 lost, to
differing extents, the right half of the
gene together with rightward-lying
chromosomal segments. The fact that an
osteosarcoma (OS-15) and a
retinoblastoma (43) lost internal portions
of this gene argued strongly that this
190-kb DNA segment, and not leftwardor
rightward-lying DNA segments, was
the repeated target of mutational
inactivation occurring during the
development of these retinoblastomas
and the osteosarcoma. (The dashed line
indicates that the deletion in tumor 43
was present in heterozygous
configuration.) (From S.H. Friend et al.,
Nature 323:643–646, 1986.)

21. LOH of chromosomal arms in CRC
High frequency of LOH allows the detection of putative TSG

23. Table 7.1 part 2 of 2 The Biology of Cancer (© Garland Science 2007)

24. Many familial cancers can be explained by inheritance of mutant TSGs.
Many familial cancers can be explained by inheritance of mutant tumor suppressor genes
These genes specify a diverse array of proteins that operate in many different intracellular sites to reduce the risk of cancer.
An anti-cancer function is the only property that is shared by these otherwise unrelated genes.
Many familial cancers can be explained by inheritance of mutant TSGs.
Inheritance of defective copies of most TSGs creates an enormously increased risk for cancer
Often a type of relatively rare tumors

25. There are two distinct classes of familial cancer genes
Gatekeepers:
Tumor suppressor genes that function to directly control the biology of cells (proliferation, differentiation, or apoptosis)
Caretakers:
The DNA maintenance genes affect cell biology only indirectly by controlling the rate at which cells accumulate mutant genes

26. Promoter methylation represents an important mechanism for inactivating tumor suppressor genes
DNA molecules can be altered covalently by the attachment of methyl groups to cytosine bases.
This modification of the genomic DNA is as important as mutation in shutting down tumor suppressor genes.
In mammalian cells, this methylation is found only when these bases are located in a position that is 5’ to guanosines, that is, in the sequence CpG.
Such methylation can affect the functioning of the DNA in this region of the chromosome.
When CpG methylation occurs in the vicinity of a gene promoter, it can cause repression of transcription of the associated gene.

27. Figure 7.18 Methylation of the
RASSF1A promoter The bisulfite
sequencing technique (Figure 7.17) has
been used here to determine the state
of methylation of the CpG island in
which the promoter of the RASSF1A
tumor suppressor gene is embedded.
Each circle indicates the site of a distinct
CpG dinucleotide in this island, whose
location within the RASSF1A promoter is
also indicated by a vertical tick line in the
map (above). Filled circles (blue) indicate
that a CpG has been found to be
methylated, while open circles indicate
that it is unmethylated. Analyses of five
DNA samples from tumor 232 indicate
methylation at almost all CpG sites in
the RASSF1A CpG island; adjacent,
ostensibly normal tissue is unmethylated
in most but not all analyses of this CpG
island. Analyses of control DNA from a
normal individual indicate the absence of
any methylation of the CpGs in this CpG
island. (These data suggest the presence
of some abnormal cells with methylated
DNA in the ostensibly normal tissue
adjacent to tumor 232.) (Courtesy of
W.A. Schulz and A.R. Fiori.
Analyses of five DNA samples from tumor 232 indicate methylation at almost all CpG sites in the RASSF1A CpG island
Adjacent, ostensibly normal tissue is unmethylated in most but not all analyses of this CpG island.
Analyses of control DNA from a normal individual indicate the absence of any methylation of the CpGs in this CpG island. 27

28. More than half of the tumor suppressor genes that are involved in familial cancer syndromes because of germ-line mutation have been found to be silenced in sporadic cancers by promoter methylation.
Ex. Rb germ line mutations  familial retinoblastoma.
Rb somatic mutations or promoter methylation  sporadic retinoblastomas.

29. The elimination of tumor suppressor gene function by promoter methylation
One copy might be methylated and the second might then be lost through a loss of heterozygosity (LOH) accompanied by a duplication of the already-methylated tumor suppressor gene copy
Both copies of a tumor suppressor gene might be methylated independently of one another
1st hit
Methylation
LOH
Methylation
(2nd hit)

30. Example: p16INK4A tumor suppressor gene
In a study of the normal bronchial (large airway) epithelia of the lungs:
p16INK4A methylation:
in 44% of (ostensibly normal) bronchial epithelial cells cultured from current and former smokers
not at all in the comparable cells prepared from those who had never smoked.
LOH in this chromosomal region:
in 71 to 73% of the two smoking populations
in 1.5 to 1.7% of never-smokers.
Conclusion: methylation of critical growth-controlling genes often occurs early in the complex, multi-step process of tumor formation, long before histological changes are apparent in a tissue

31. 31

32. Example: the BRCA1 gene:
Silencing of genes through promoter methylation can also involve the “caretaker” genes
Example: the BRCA1 gene:
Its product  maintaining the chromosomal DNA in ways that are still poorly understood.
Inheritance of mutant alleles of BRCA1  a high lifetime risk of familial breast and, to a lesser extent, ovarian cancer syndrome.
10 to 15% of sporadic breast carcinomas carry inactive BRCA1 gene copies that have been silenced through promoter methylation.

33. Promoter methylation is common during the development of a wide variety of human cancers
the frequency of methylation of a specific gene varies dramatically from one type of tumor to the next.
Tumor suppressor genes as well as caretaker genes undergo hypermethylation.
Perhaps the 33

34. The (Neurofibromin ) NF1 protein acts as a negative regulator of Ras signaling
When cells stimulated by growth factor, they may degrade NF1, enabling Ras signaling to proceed without interference by NF1.
After 60 to 90 minutes, NF1 levels return to normal, and the NF1 protein that accumulates helps to shut down further Ras signaling in a form of negative-feedback control
Figure 7.22 Neurofibromin and the
Ras signaling cycle As illustrated in
Figure 5.30, the Ras protein passes
through a cycle in which it becomes
activated by a guanine nucleotide
exchange factor (GEF), such as Sos, and
becomes inactivated by a GTPaseactivating
protein (a Ras-GAP). One of
the main Ras-GAPs is neurofibromin
(NF1), the product of the NF1 gene.
Interaction of Ras with NF1 can increase
the GTPase activity of Ras more than
1000-fold. The structure of the domain
of NF1 that interacts with Ras is
illustrated here. A subdomain of NF1
termed the “arginine finger” (top)
carries a critical arginine (R1276) that is
inserted into the GTPase cleft of Ras and
actively contributes to the hydrolysis of
GTP to GDP by Ras. Mutant forms of
NF1 (carrying amino acid substitutions)
observed in neurofibromatosis patients
are indicated by light gray spheres that
are labeled by gray boxes; experimentally
created mutations have also generated a
number of amino acid substitutions
(dark gray spheres) that also compromise
NF1 GAP function. In addition, a largescale
deletion (D53) and an insertion
(“Type II insert”) found in patients are
shown; both are also disease-associated.
(From K. Scheffzek et al., EMBO J.
17:4313–4327, 1998 34

35. Neurofibromatosis type 1 is a relatively common familial cancer syndrome, with 1 in 3500 individuals affected on average worldwide.
The primary feature of this disease is the development of benign neurofibromas of the cell sheaths around nerves in the peripheral nervous system
On occasion, a subclass of these neurofibromas, progress to malignant tumors termed neurofibrosarcomas.

36. In neuroectodermal cells lacking NF1 function, Ras proteins are predicted to exist in their activated, GTP-bound state for longer than- normal periods of times.
In fact, in the cells of neurofibromas, which are genetically NF1–/–, elevated levels of activated Ras and Ras effector proteins can be found
Consequently, the loss of NF1 function in a cell can mimic functionally the activated Ras proteins that are created by mutant ras oncogenes.